Supercooling Pretreatment Improves the Shelf-Life of Freeze-Dried Leuconostoc mesenteroides WiKim32

Storage stability of freeze-dried lactic acid bacteria is a critical factor for their cost-effectiveness. Long-term storage of lactic acid bacteria enables microbial industry to reduce distribution costs. Herein, we investigated the effect of cold adaptation under supercooling conditions at −5°C on the viability of Leuconostoc mesenteroides WiKim32 during the freeze-drying process and subsequent storage. Cold adaptation increased the thickness of exopolysaccharides (EPS) and improved the viability of freeze-dried Leu. mesenteroides WiKim32. Compared to non-adapted cells, cold-adapted cells showed a 35.4% increase in EPS thickness under supercooling conditions. The viability of EPS-hydrolyzed cells was lower than that of untreated cells, implying that EPS plays a role in protection during the freeze-drying process. Cold adaptation increased the storage stability of freeze-dried Leu. mesenteroides WiKim32. Fifty-six days after storage, the highest viability (71.3%) was achieved with cold adaptation at −5°C. When EPS-containing broth was added prior to the freeze-drying process, the viability further increased to 82.7%. These results imply that cold adaptation by supercooling pretreatment would be a good strategy for the long-term storage of Leu. mesenteroides WiKim32.


Introduction
Sensory characteristics of fermented vegetable foods, such as kimchi, pao cai, and sauerkraut, can be enhanced using Leuconostoc spp. Leu. mesenteroides WiKim32, isolated from kimchi at an early stage of fermentation, has been used as a kimchi starter owing to its advantageous traits; particularly, it extends the shelf-life and improves the sensory properties of kimchi [14]. Our previous study revealed that supercooling pretreatment at −5°C improves the storage stability of Lac. brevis WiKim0069 [13]. The overall objective of this study was to examine the effects of cold adaptation on exopolysaccharides (EPS) production and cell wall composition, which improve the viability of Leu. mesenteroides WiKim32. Furthermore, the effect of adding various concentrations of EPS before the freeze-drying process on cell viability was investigated.

Bacterial strain and Culture Conditions
Leu. mesenteroides WiKim32 (KFCC11639P), isolated from kimchi and developed as a kimchi starter, was obtained from the culture collection at the World Institute of Kimchi (Gwangju, Republic of Korea). Stock cultures were stored at −80°C (MDF4V; Panasonic, Japan) in De Man, Rogosa, and Sharpe (MRS) medium (Difco Laboratories, USA) with 20% glycerol. Working cultures were grown in MRS medium at 30°C for 24 h.

Cold Adaption Procedure and Cell Viability
The cold adaptation process and measurement of cell viability were carried out using the procedure described by Choi et al. [13]. Leu. mesenteroides WiKim32 was grown in MRS medium at 30°C for 24 h and harvested by centrifugation (10,000 ×g at 25°C for 20 min). The extra MRS broth was stored at 4°C as a suspension solution for subsequent experiments. The precipitated cells collected by centrifugation were washed twice with sterile phosphate-buffered saline (3M Company, USA) and concentrated to 4 × 10 10 CFU/ml in either saline or the 24 hcultured MRS broth. The final cell concentration was adjusted to 2 × 10 10 CFU/ml with a uniform volume of sterile 20% solution of soy powder as cryoprotectant. The cells suffered from cold stress for 2 h at 10°C or −5°C prior to freeze-drying. A control sample was not exposed to cold stress. The freeze-drying process was conducted at −80°C with a vacuum degree lower than 1 Pa using a freeze dryer (FDCF-12012; Operon, Republic of Korea). The freezedried cells were separately prepared for each storage period and kept for 56 days at −20°C.
Cell viability was evaluated using the plate count method instantly after freeze-drying and during storage periods. The appropriate diluent was poured onto MRS agar and incubated at 30°C for 48 h. Bacterial colonies were counted and calculated as CFU/ml.

Preparation of EPS and EPS-Free Bacteria
EPS purification. The EPS was purified following the procedure reported by Choi et al. [15], with minor adjustments. After cell harvesting, discarded MRS broth was boiled at 100°C for 5 min for inactivated enzymes and the supernatants were obtained by centrifugation (10,000 ×g for 20 min at 4°C). For ethanol precipitation and lyophilization, three times the amount of 95% ethanol was added to the supernatants and kept at −20°C overnight before centrifugation (10,000 ×g for 20 min at 4°C). The protein was removed by dissolving it in distilled water and deproteinizing it with 4% trichloroacetic acid (Sigma-Aldrich, USA). Crude EPS was reprecipitated with 95% ethanol, dissolved in distilled water, dialyzed with distilled water for 2 days at 4°C using Slide-A-Lyzer dialysis cassettes (10K MWCO; Thermo Scientific, USA), and dried at −20°C overnight. Purified EPS was added to the cell viability test as a cryoprotectant.
Enzymatic hydrolysis of EPS. To reduce the EPScontent of Leu. mesenteroides Wikim32, bacterial cells were treated with hydrolysis enzyme, Dextranase Plus L (Novozymes, Chaetomium erraticum). Subsequently, 500 μl of Dextranase solution was added to 200 ml of bacterial culture broth. Enzymatic hydrolysis was performed for 12 h at 30°C. The EPS-hydrolyzed cells were harvested by centrifugation at 10,000 ×g for 20 min at 4°C and washed with distilled water three times. The viability of EPS-hydrolyzed cells was measured with or without 0.1% purified EPS as a cryoprotectant using the plate count method.

Monosugar Composition
Monosaccharide compositions were analyzed following the method described by Choi et al. [15]. The neutral sugars of cold-adapted and non-adapted cells was acidic hydrolyzed, converted into their corresponding alditol acetates, and measured using a gas chromatography apparatus (7890A; Agilent Technology, USA) equipped with a flame ionization detector. A capillary column (DB-225; 30 m length, 0.25 mm diameter, 0.25 μm film thickness; Agilent Technology), wherein the temperature was controlled at 100-220°C with an increasing rate of 5°C/min, was used.

Transmission Electron Microscopy (TEM)
The morphological changes of cold-adapted cells were analyzed using a transmission electron microscope (JEM-1400; Jeol, Japan). Prior to observation, each sample fixed in 2% glutaraldehyde (Merck, Germany) was post-fixed overnight in 1% osmium tetroxide (Sigma-Aldrich), dehydrated with a graded ethanol series of 30%, 50%, and 70%, embedded in LR white acrylic resin (Sigma-Aldrich) at 50°C for 24 h, and then sectioned using an ultra-microtome with a diamond knife. The sections were placed on the center of a TEM grid and stained with uranyl acetate (Sigma-Aldrich) and lead citrate (Sigma-Aldrich). EPS thickness was examined using DigitalMicrograph software (Gatan Inc., USA). The average diameter was calculated from the values for 16 bacteria taken at random locations of each cell.

Statistical Analysis
All experiments were carried out three independent experiments. All data were analyzed by analysis of variance using Statistical Package for the Social Sciences software (version 19.0; IBM Corp., USA). Mean values were analyzed using Duncan's multiple tests to determine significant differences in the treatments at p < 0.05.

Effect of cold adaption on EPS thickness of Leu. mesenteroides WiKim32
Structural changes in bacterial cell walls were determined using transmission electron microscopy (TEM) (Fig. 1). EPS thickness of Leu. mesenteroides WiKim32 varied with pretreatment conditions (F = 62.4, df = 2,45, p < 0.001). The thickest EPS (51.6 ± 3.52 nm) was observed in cold-adapted cells at −5°C, followed by those in coldadapted cells at 10°C (42.0 ± 3.34 nm; Table 1). Compared with that of non-adapted cells, EPS thickness of coldadapted cells increased by 10.2-35.4%. Monosugar composition strongly supported the reason EPS thickness depended on adapted conditions. Carbohydrates in non-adapted cells are mainly composed of arabinose (1.6%), galactose (2.8%), and glucose (8.7%) ( Table 2). After cells were adapted at −5°C, carbohydrate contents were changed to arabinose (1.7%), galactose (2.9%), and glucose (9.6%). There are four structural models for cell walls   of LAB: (a) cell wall composed of the outer layer, peptidoglycan, and lipoteichoic acids; (b) cell wall enveloped by EPS; (c) cell wall surrounded by the surface layer protein (SLP); (d) cell wall crosslinked EPS and SLP [13]. Among these models, Leu. mesenteroides WiKim32 has a cell wall model in which EPS is attached to the cell wall [15]. In this study, supercooling pretreatment further increased the EPS thickness of Leu. mesenteroides WiKim32.

Effect of Enzymatic Hydrolysis of EPS on Cell Viability
To understand the role of EPS in the viability of Leu. mesenteroides WiKim32, EPS-hydrolyzed cells were manufactured (Fig. 2). The glucose content was reduced by hydrolysis of EPS attached to the cell wall, suggesting that EPS was successfully removed ( Table 2). Significant difference in viability was observed with cell type (F = 173.0, df = 1,12, p < 0.001) (Fig. 3). Viability of EPS-hydrolyzed cells was lower than that of untreated cells, suggesting that EPS plays a role in protection during the freeze-drying process. The viability of LAB is severely affected by protective agents including EPS and soy powder. Regardless of cell types, the least viability was recorded with the absence of protective agents (F = 2927.5, df = 2,12, p < 0.001). With EPS as a protective agent, the viability of both cell types increased, showing 18.6% and 12.1% for untreated cells and EPS-hydrolyzed cells, respectively. The highest viability of 89.6% was obtained in untreated cells with soy powder as a protective agent, followed by 66% in EPS-hydrolyzed cells with soy powder.
Microbial EPS are extracellular carbohydrate polysaccharides that are not permanently attached to the cell surface. EPS produced by LAB has shown various health-benefit properties such as anticancer, antioxidant, antiviral, immunostimulant, and cholesterol-lowering effects [15,16]. They also aid in the protection of Leu. mesenteroides WiKim32 against environmental stress such as dehydration, unfavorable temperature, osmotic stress, and antibiotics [17]. In our study, the removal of EPS resulted in low viability, indicating that EPS layer surrounding the cell surface showed protective activity against the freeze-drying process. Considering that the addition of a few EPS prior to freeze-drying improved the viability of Leu. mesenteroides WiKim32, moreover, EPS might be used as a protective agent.

Effect of Cold Adaption by Supercooling Pretreatment on Stability
The storage stability of freeze-dried Leu. mesenteroides WiKim32 cells varied with pretreatment conditions (F =  (Fig. 4). Cold-adapted cells had higher viability than non-adapted cells during freeze-drying and following storage. The viability of cold-adapted cells was maintained at more than 80% after freeze-drying, while non-adapted cells had a significantly reduced viability of 67.2%. Among pretreatment methods, cold adaptation at −5°C was best for viability improvement, showing 86.8% after 28 days of storage. The viability of freeze-dried Leu. mesenteroides WiKim32 cells differed from the type of suspension solution including sterile saline and MRS broth cultured for 24 h. Stability of Leu. mesenteroides WiKim32 cells cultured in MRS broth containing 0.1% (w/v) EPS were higher than that in saline. The highest viability of 82.7% was maintained for up to 56 days in the 24-h cultured MRS broth for cold-adapted Leu. mesenteroides WiKim32 at −5°C, followed by coldadapted cells at 10°C. In our previous studies, cold adaptation has the advantage of the shelf-life extension of freeze-dried Lactobacillus brevis WiKim0069 [12,13]. Supercooling condition which is below the freezing point further increased the resistance of Lac. brevis WiKim0069 to freeze-drying, leading to higher viability than cold adaptation at 10°C [13]. Both cation influx and SLP production increased when cells were exposed to adaptation conditions. Intracellular cations such as Ca 2+ and Mg 2+ can induce the activation of calcineurin, which is known as calcium-dependent serine-threonine phosphatase, and resistance to osmotic stress [18][19][20][21]. SLP formation resulting from cold adaptation made the cell surface thicker to protect cells against freeze-drying. Surface-layer thickness depended on cold adaptation conditions. Likewise, EPS plays an important role in the adherence of cells and provides a protective barrier for cells. In the presence of 0.1% (w/v) EPS, the viability of Leu. mesenteroides WiKim32 cells increased more than 10%. Our results are consistent with a previous study by Kim et al. [22], indicating that the presence of EPS produced by Pseudoaltermonas elyakovii improved the resistance of Escherichia coli against freeze-thaw cycles. The presence of EPS helps Leu. mesenteroides WiKim32 to resist cold stress, suggesting that it could be used as a cryoprotective agent. Cold adaptation by supercooling pretreatment would be a good strategy for the long-term storage of lactic acid bacteria such as Leuconostoc spp. and Lactobacillus spp.